Deferoxamine B, represented by formula I, is a polyhydroxamate iron chelator that is useful for reducing iron concentration in human blood plasma. 
The systematic chemical name of deferoxamine B (also known as deferriferrioxamine B) is N′-[5-[[4-[[5-(acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxy-butanediamide. Deferoxamine B has the desirable property of high affinity for ferric iron (Ka=1031) coupled with a very low affinity for calcium (Ka=102). Goodman and Gilman, The Pharmacological Basis of Therapeutics 1668 (9th ed. 1996).
Deferoxamine B is indicated for treatment of acute iron intoxication and chronic iron overload due to transfusion dependant anemias. It promotes iron excretion in patients with secondary iron overload from multiple transfusions, as may occur with treatment of some chronic anemias such as thalassemia. Long term therapy slows accumulation of hepatic iron and retards or eliminates progression of hepatic fibrosis. Physicians Desk Reference 2010 (54th ed. 1999). Deferoxamine B is not well-absorbed orally; it must be administered parenterally.
Industrial scale fermentation processes for producing deferoxamine B use the Streptomyces pilosus bacteria strain, which produces a variety of polyhydroxamate compounds, but predominantly deferoxamine B, in a culture medium poor in iron. Belgian Patent No. 619,532. Deferoxamine B is typically isolated from the fermentation broth as its hydrochloride salt. The hydrochloride salt is not pharmaceutically acceptable for parenteral administration to humans. Therefore, it must be converted into a pharmaceutically acceptable salt. The mesylate salt is the FDA approved deferoxamine salt. The U.S. Pharmacopeia & National Formulary directs that pharmaceutical grade deferoxamine B mesylate contain not more than 120 ppm chloride. USP/NF 24/19, pp. 499-500 (1999). This has proven to be a challenging standard to meet and there remains a need for an improved process for transforming deferoxamine B produced by fermentation into a pharmaceutically acceptable pure mesylate salt for administration to patients.
Belgian patent 619,532 discloses purification of deferoxamine B obtained via fermentation using adsorption chromatography. Activated carbon, activated diatomaceous earth (e.g. fuller's earth) or ion-exchange resin (Asmit) are recommended adsorbents. Alternative adsorbents are said to include aluminum oxide, magnesium silicates, silica gel, gypsum and ion-exchange resins. According to the Belgian 619,532 patent, deferoxamine B may be eluted using a mobile phase of methanol-water, pyridine-water or acetic acid-methanol.
International Publication No. WO 93/09088 and European patent 347,163 describe chromatography over silica gel as a method for purifying deferoxamine B produced by synthetic means, not by microbiological means.
International Publication No. WO 93/03045 describes purification of iron chelate complexes of deferoxamine B and structurally related compounds using a polystyrene adsorption resin.
U.S. Pat. Nos. 3,153,621 and 3,118,823 disclose partial purification of deferoxamine B using ion-exchange resins. It is believed that the teachings of these patents lead to a mixture of deferoxamine B with other polyhydroxamates that are produced by the Streptomyces pilosus strain.
Belgian Patent 616,139 discloses that deferoxamine B mesylate salt can be obtained from the deferoxamine B hydrochloride salt by passing an aqueous solution of deferoxamine hydrochloride over Dowex-1, X-16 anion exchange resin (in OH− form), adding methanesulfonic acid in equivalent quantity to the resulting deferoxamine base in aqueous solution, then evaporating the water and, lastly, purifying the deferoxamine mesylate salt by recrystallization from aqueous alcohol or a mixture of water-methanol-acetone.
U.S. Pat. No. 5,374,771 describes purification of crude deferoxamine B hydrochloride by ion-exchange chromatography and multiple recrystallizations. The mesylate salt is prepared directly from the purified deferoxamine B hydrochloride by contacting with an anion exchange resin having mesylate counter-ion. Deferoxamine B mesylate is obtained from the aqueous solution by lyophilization.
Bickel, H. et al., Helvetica Chimica Acta, 1385-1389 (1963), describes purification of deferoxamine B base by multiple recrystallizations from a water-alcohol mixture. The deferoxamine B base is prepared by anion exchange, evaporation to dryness, and multiple recrystallizations. The deferoxamine B base is then suspended in a water-methanol mixture and a mineral acid salt prepared. Subsequently the deferoxamine B solution is evaporated and the residue is recrystallized from a water-methanol mixture.
Removal of chloride ion from an aqueous solution of deferoxamine B is one of the steps in each of the methods described above for purifying deferoxamine B from a fermentation broth. In each case, the chloride ion is removed using an anion exchange resin. However, ion-exchange resins alone are not effective for isolating deferoxamine B mesylate free of chloride ion as is required in order to achieve a pharmaceutically acceptable state of purity.
The above-mentioned purification methods that use silica gel or aluminum oxide as adsorbents are inefficient, time consuming and expensive. The other conventional substitutes—activated charcoal, diatomaceous earth, magnesium silicates, and gypsum—are poor adsorbents of deferoxamine. For these reasons, there remains a need in the art for a rapid, efficient and inexpensive method for obtaining deferoxamine B mesylate from a fermentation broth free of chloride ion, or at least free of chloride anion in an amount greater than 120 ppm.
Furthermore, the above-described methods do not efficiently remove fermentation products that are structurally related to deferoxamine B, which must be removed before use of the deferoxamine B in a pharmaceutical product. Typically, an extract from a deferoxamine-producing fermentation broth contains, relative to the deferoxamine B content, approximately 6 to 20 mole % polyhydroxamate compounds that are structurally related to deferoxamine B. Such compounds include other deferoxamines, such as deferoxamine A, C, D1, D2, E, F and G. Since deferoxamine B and the other deferoxamines have similar chemical properties, none of the known purification processes or combinations thereof have been able to reduce the quantity of the impurities below about 2.5%.
In addition to their uncertain therapeutic, and possibly toxic effect it is important to remove these impurities in order to accurately determine the deferoxamine B concentration in a solution, such as a sterile solution for injection. The USP/NF specifies that a deferoxamine B injection solution contain between 90.0 and 110% of the labeled concentration. USP/NF 24/19, p. 500 (1999) The assay for determining concentration specified by the USP/NF is a photometric absorbence intensity measurement of the iron complex at 485 nm. Id. The structurally related impurities also form complexes which absorb in the 485 nm range, leading to an overestimation of the deferoxamine B content. We have found that a photometric assay of a solution of deferoxamine B mesylate obtained by fermentation according to methods known in the art overestimates the concentration of deferoxamine B by about 3%. Thus, there remains a need in the art for a rapid, efficient and inexpensive method for obtaining deferoxamine B mesylate from a fermentation broth free of other polyhydroxamates formed by the metabolic processes of Streptomyces (e.g. pilosus or 101/87) as well as free of chloride ion.